1. Field of the Invention
The present invention relates to a fluorescence imaging device, and more particularly, to a imaging device based on a fluorescence microscope optical system. More particularly, the present invention relates to a fluorescence imaging device configured to be capable of observing fluorescent images of subjects dyed with different fluorophores, which has a simple structure and is capable of being operated easily.
2. Description of the Prior Art
When ultraviolet rays and visible rays with a short wavelength are irradiated to a specimen, dye molecules of the specimen emit a light. A microscope used for observing a fluorescence of a visible ray range which is emitted at that time is referred to as a fluorescence microscope.
The specimen of the fluorescence microscope should have an element that develops a fluorescence by itself or emits a fluorescence when it absorbs a short wavelength. For this purpose, the specimen is processed by a fluorophore (a fluorescent dye) and a light, of which a wavelength is absorbed to the fluorophore, is irradiated to the specimen so as to observe the specimen through a radiation light emitted from the specimen.
The fluorescence microscope may easily sense a very small amount of a fluorophore. Thus, the fluorescence microscope is used when studying a distribution or a moving path of a fluorophore existing in a specimen, a cell, etc. which may not be identified by a human.
In addition, such a fluorescence microscope has been used for various types of imaging devices which may automatically analyze a feature of a nucleic acid, an intracellular material, or a cell itself. For example, an automatic cell counter has been frequently used in a state where a bright-field optical system is incorporated therein. When a specimen containing a cell to be measured is not purely separated, the bright-field optical system cannot provide a correctly measured value. In order to overcome this problem, automated fluorescence cell counters have been developed which measure a fluorescence dyed specimen using a fluorescence microscope method. In addition, an equipment such as a DNA sequencer, a DNA chip scanner, or an image cytometry is also configured to basically incorporate a fluorescence microscope optical system and to connect the fluorescence microscope system with a proper driving unit and software so as to acquire and analyze a fluorescence image.
The configurations and functional actions of conventional fluorescence microscopes are as follows.
A conventional fluorescence microscope selects a monochromatic light which coincides with an absorption wavelength of a fluorescent body in a white light through an excitation filter, adjusts the path of the monochromatic light of the selected absorption wavelength using a dichroic mirror so as to irradiate the monochromatic light to the specimen through an objective lens, selects a light which coincides with a color development wavelength of the fluorescent body of the specimen in the light produced by the fluorescent body of the specimen and transmitted by the objective lens and the dichroic mirror, using an emission filter, and provides the selected light to an image sensor.
The image sensor is implemented by an imaging element such as an eyepiece or a Charge Coupled Device (CCD) and detects and presents a color development wavelength of the fluorescent body attached to the specimen so that the shape of the specimen can be observed.
Recently, fluorescence microscopes of a type configured to irradiate various lights to a specimen to obtain fluorescent images and then compare the fluorescent images with each other so as to observe a correct shape of the specimen, rather than being configured to obtain a single fluorescent image according a light irradiated to a specimen, are being developed. Schematic configurations of such fluorescence microscopes are illustrated in FIGS. 1 and 2.
The fluorescence microscope illustrated in FIG. 1 is adapted to use a separate light source for each wavelength and to execute an observation while changing individual filter assemblies 100 (including a light sources 101, a focusing lens 102, an excitation filter 103, a dichroic mirror 104, and an emission filter 105) as desired.
However, the fluorescence microscope illustrated in FIG. 1 has a problem in that, since the light source 101 is irradiated via an objective lens 107, a subject S is distant from the light source 101 and thus, the intensity of radiation is weak so that the intensity of an observed fluorescence signal is weakened.
The fluorescence microscope of this type should be provided with individual filter assemblies 100. Thus, the fluorescence microscope has a complicated configuration and a large volume, which inevitably increases the manufacturing costs.
The fluorescence microscope illustrated in FIG. 2 is configured to be provided with one light source 101 configured to irradiate excitation light and one excitation filter 102 to detect lights of various wavelengths using a plurality of dichroic mirrors 104 and image sensors 106.
The fluorescence microscope of the type illustrated in FIG. 2 has a stable structure since it is not required to move a filter assembly unlike the fluorescence microscope illustrated in FIG. 1. However, the fluorescence microscope illustrated in FIG. 2 also has a problem in that, since the light source 101 irradiates the subject S via the objective lens 107, the intensity of radiation is weak and thus, the intensity of a fluorescence signal is weakened. In addition, since it is necessary to use a plurality of dichroic mirrors 104 and expensive image sensors 106, the manufacturing costs are also increased.
Further, the fluorescence microscopes illustrated in FIGS. 1 and 2 are provided with dichroic mirrors 104 between an objective lens 107 and an image sensor 106 and, when observing a bright-field image, the dichroic mirrors 104 should be removed. However, the light path when the dichroic mirrors 104 are present and the light path when the dichroic mirrors 104 are absent become different from each other due to the refraction of light, and a bright-field image and a fluorescence image become substantially different from each other without being overlapped. Consequently, there is a problem in that it is difficult to compare the two images.
In order to solve this problem, an infinity-corrected objective lens is used instead of a finite conjugate objective lens. In such a case, there is a problem in that, since a tube lens having a predetermined focal length is additionally required, more space is required and the size of the equipment is increased.